Betalain producing cell cultures ofBeta vulgaris L. var. bikores monogerm (red beet)

Summary The betalains are a class of natural pigments comprising the yellow betaxanthins and the violet betacyanins. Callus lines developed fromBeta vulgaris, L. var. bikores monogerm exhibited cell colors ranging from white/green (nonpigmented) through yellow, orange, red, and violet and were representative of all betalain pigments found in the whole plant. The betalains have gained particular interest from the food industry as potential natural alternatives to synthetic food colorants in use today. Red beet extracts (E162), which contain significant amounts of the betacyanins, are currently used in products such as yogurts and ice creams. We describe here the characteristics of culture growth and betalain production for cell suspensions derived from the orange (predominantly betaxanthin-producing) and violet (betacyanin producing) callus lines. The major factors affecting betalain biosynthesis in both cultured and whole plant tissues are reviewed. Presented in the Session-in-Depth Batch Production and Fermentation at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–29, 1991.     

Relationship between type and age of the inoculum cultures and betalains biosynthesis by Beta vulgaris hairy root culture

From a study of the relationship between the type and age of the inocula, and the growth and biosynthesis of betalains in a Beta vulgaris hairy root culture, the best results were achieved with a 14 d inoculum grown in submerged culture giving 42 mg betalains (16 mg betacyanins and 26 betaxanthins) and 1.5 g dry biomass in 40 ml medium.     

Betacyanins and phenolic compounds from Amaranthus spinosus L. and Boerhavia erecta L

Stem bark extracts of Boerhavia erecta L. (erect spiderling) and Amaranthus spinosus L. (spiny amaranth), two wild growing weed plants used in traditional African medicine, were characterized with respect to their phenolic profile including the betalains. While the main betalains in A. spinosus were identified as amaranthine and isoamaranthine, the major betacyanins in B. erecta were betanin, isobetanin together with neobetanin. The latter showed higher betalain concentrations amounting to 186 mg/100 g, while the former contained 24 mg betacyanins in 100 g of the ground plant material. Extracts of A. spinosus were found to contain hydroxycinnamates, quercetin and kaempferol glycosides, whereas catechins, procyanidins and quercetin, kaempferol and isorhamnetin glycosides were detected in B. erecta. The amounts of these compounds ranged from 305 mg/100 g for A. spinosus to 329 mg/100 g for B. erecta.     

Production of betalains by suspension cultures of Chenopodium rubrum L.

Red-violet cell suspension cultures of Chenopodium rubrum were found to accumulate the betacyanins amaranthin, celosianin and betanin and the betaxanthins vulgaxanthin I and vulgaxanthin II. Under a 16-h daylight regime the cells accumulated 0.3–0.4% betacyanins on a dry mass basis after 2–3 weeks of cultivation on the growth medium. Experiments to define a production medium for betacyanins failed with this habituated line. The accumulation could however be increased up to 1% or 100 mg betacyanins/1 by feeding tyrosine and by adaptation of the inoculum size to the nutrient concentration.     

Bioreactor for cultivation of red beet hairy roots and in situ recovery of primary and secondary metabolites

To arrive at an appropriate bioreactor design and in situ recovery of the products, red beet hairy roots were used as a model system where the levels of betalain pigments (betacyanins and betaxanthins) were followed as secondary metabolite and the peroxidase enzyme as primary metabolite. Medium volume and other kinetic parameters were found to play significant roles by way of directly affecting the biomass yield rather than a specific metabolite. The hydrodynamic stress created on the roots by large culture volume could be minimized by pulse‐feeding of medium in shake‐flasks; and by separating the biomass chamber from the aerated medium reservoir in circulatory fed‐batch bioreactor. Accordingly the bioreactor was modified to provide anchorage and air‐enrichment chamber which resulted in higher formation of both the metabolites than in shake‐flasks. Various down‐stream processing aspects such as in situ release of pigments by non‐destructive methods, followed by adsorption through a column and recovery by desorption were optimized for betalains. A strategy for simultaneous recovery of pigment and peroxidase was worked out using aqueous two phase extraction (ATPE).     

Betalains in the era of global agri-food science, technology and nutritional health

Natural pigments from plants are of growing interest as substitutes for synthetic dyes in the food and pharmaceutical industry and they increase their added value if they possess positive effects on health. These pigments can be added as such if they are in the legal authorized lists of additives or can be added as phytochemical-enriched plant extract achieving the original product, which has received it, the new nomenclature of functional food. In this way, we comprise on this review a wide point of view of a group of natural pigments known as betalains. From a chemical point of view, betalains are ammonium conjugates of betalamic acid with cyclo-DOPA (betacyanins, violet) and aminoacids or amines (betaxanthins, orange or yellow), which are compounds present in our diet. Besides and taking into account that one type of betalain, betanin is approved as food colorant (E-162) by the European Union and that enlarges the specific weight of these compounds in the diet, we have evolved an overview from the biosynthesis, technology and promoting production, industrial uses as pigments up to physiological and nutritional biovailability or biological and health-promoting properties of betalains for accessible information to industrials, researchers and consumers.     

Additional betalain accumulation by genetic engineering leads to a novel flower color in lisianthus (Eustoma grandiflorum)

Betalains, comprising violet betacyanins and yellow betaxanthins, are pigments found in plants belonging to the order Caryophyllales. In this study, we induced the accumulation of betalains in ornamental lisianthus (Eustoma grandiflorum) by genetic engineering. Three betalain biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in lisianthus, in which anthocyanin pigments are responsible for the pink flower color. During the selection process on hygromycin-containing media, some shoots with red leaves were obtained. However, most red-colored shoots were suppressed root induction and incapable of further growth. Only clone #1 successfully acclimatized and bloomed, producing pinkish-red flowers, with a slightly greater intensity of red color than that in wild-type flowers. T₁ plants derived from clone #1 segregated into five typical flower color phenotypes: wine red, bright pink, pale pink, pale yellow, and salmon pink. Among these, line #1-1 showed high expression levels of all three transgenes and exhibited a novel wine-red flower color. In the flower petals of line #1-1, abundant betacyanins and low-level betaxanthins were coexistent with anthocyanins. In other lines, differences in the relative accumulation of betalain and anthocyanin pigments resulted in flower color variations, as described above. Thus, this study is the first to successfully produce novel flower color varieties in ornamental plants by controlling betalain accumulation through genetic engineering.     

Qualitative and quantitative aspects of betalains biosynthesis in Amaranthus caudatus L. var. pendula seedlings

Seedlings of Amaranthus caudatus L. var. Pendula were used to study the influence of several treatment: white light, 3,4-dihydroxyphenylalanine (DOPA), kinetin, gibberellic acid (GA₃) on betalains biosynthesis. The pigments, betacyanins and betaxanthins, were separated using a Sephadex G-15 column chromatography. Qualitative as well as quantitative differences were observed according to the treatments applied.The amaranthin biosynthesis seemed to be favored in the absence of DOPA. Under the combined effect of kinetin and white light a small quantity of betanin was also synthesized. Adding exogenous DOPA led to a more diversified production which included betacyanins (amaranthin and betanin), betaxanthins (vulgaxanthin and miraxanthin), and even dopachrome. As a general rule, kinetin activated the betalains biosynthesis whereas GA₃ inhibited it. The stimulating effect of white light was always much greater than that of kinetin.Abbreviations GA₃ gibberellic acid - GA₄ gibberellin 4 - BHT 2,6-diter-butyl-4-methylphenol - DOPA 3,4-dihydroxyphenylalanine - Kinetin 6-furfurylaminopurine     

Structural implications on color, fluorescence, and antiradical activity in betalains

Betalains are water-soluble pigments with high antiradical capacity which bestow bright colors on flowers and fruits of most plants of the order Caryophyllales. They are classified as betacyanins, exhibiting a violet coloration, and betaxanthins, which exhibit yellow coloration. Traditionally, betalains have been defined as condensation products of betalamic acid with different amines and amino acids, but the implication of the pigment structure for their properties has not been investigated. This paper explores different structural features of the betalains, revealing the clues for the switch from yellow to violet color, and the loss of fluorescence. A relevant series of 15 betalain-related compounds (both natural and novel semisynthetic ones) is obtained and characterized by chromatography, UV-vis spectrophotometry, fluorescence, and electrospray ionization mass spectroscopy. Antiradical properties of individual pure compounds in a broad pH range are studied under the ABTS^•+ radical assay. Relevance of specific bonds is studied, and differences between betaxanthins and betacyanins are used to explore in depth the structure–antiradical activity relationships in betalains.     

Linkage among the R, Y and BI loci in table beet

The primary pigments in table beet are the betalains, which are comprised of the red-violet betacyanins and the yellow betaxanthins. The presence of dominant alleles at two linked loci (R and Y) condition the qualitative production of betalain pigment in the beet plant. Red-pigmented roots are observed only in the presence of dominant alleles at both the R and Y loci, while white roots are conditioned by recessive alleles at the Y locus, and yellow roots by the genotype rrY-. A newly described gene ’blotchy’ (bl) conditions a blotchy or irregular pigment patterning in either red or yellow roots. The objective of the present investigation was to characterize the linkage relationships between the R and Y lociand the bl gene by evaluating segregating progenies developed from a series of matings of colored and white table beets. Due to epistatic interactions among the R, Y, and bl loci, algorithms for estimating linkage were developed using maximum-likelihood estimators for each cross. The two-point linkage estimate between the R and Y loci pooled over eight crosses was 7.4±1.7 cM. Segregation data indicated the bl gene is linked to the R and Y loci.The recombination fraction between R and bl was estimated from a pooled sample of four crosses at 16.7±10.8 cM. The most-likely gene order was R-Y-bl. These data demonstrate that the bl gene is a third locus conditioning betalain pigment production in table beet. The R-Y-bl genomic region is therefore important in the genetic control of betalain biosynthesis in table beet. Received: 18 May 1999 / Accepted: 29 August 1999     

甜菜色素的生物学研究进展

甜菜色素(Betalains)是一类以六碳结构为骨架的水溶性色素,存在于石竹目Caryophyllales的绝大部分科和一类真菌中。目前已知甜菜色素大约有75种,都属于季胺型生物碱,可以分为两类:甜菜黄素(Betaxanthin)和甜菜红素(Betacyanin)。甜菜色素除了赋予花、果、叶着色,吸引昆虫,提高植物本身抗逆能力等外,也具有优良的抗氧化作用。甜菜色素的合成代谢始于酪氨酸,是由4~5个关键酶催化反应和一系列自发反应构成的反应网络。本文结合国内外最新研究进展,对甜菜色素理化性质、生理功能、合成以及应用做较为全面的综述。     

Scaled-up biotechnological production of individual betalains in a microbial system

The recent interest in plant pigment betalains as bioactive compounds and chemopreventive agents has led to the search for a reliable and scalable process to obtain them. The cloning of the novel and efficient enzyme 4,5-DOPA-extradiol dioxygenase from Gluconacetobacter diazotrophicus in an expression vector, and the subsequent heterologous expression in Escherichia coli cultures has led to the start-up of a biotechnological production system of individual pigments. The aim of this study was to search for the optimal conditions for the production of betalamic acid in microbial factories and the scaled-up obtention of the derived pigments. Four different betaxanthins and two betacyanins were obtained after the addition of non-transformable amines and amino acids and their condensation with the betalamic acid produced by the dioxygenase. The scaled-up obtention and purification of betalains improved the yields of the previous methodologies reaching quantities by up to 150 mg of pure compounds.     

Beetroot betalain inhibits peroxynitrite-mediated tyrosine nitration and DNA strand cleavage

Two major betalains, red-purple betacyanins and yellow betaxanthins, were isolated from red beetroots (Beta vulgaris L.), and their peroxynitrite (ONOO(-)) scavenging capacity was investigated. Apparent colours of the betalains were bleached by the addition of ONOO(-), and the absorbance decreases were suppressed in the presence of glutathione, a ONOO(-) scavenger. After bleaching, a new absorption maximum was observed at 350 nm in the spectrum of the resulting reaction mixture. New peaks were detected from HPLC analysis of the reaction products of betanin, a representative constituent of red beetroot betacyanins, treated with ONOO(-) monitoring at 350 nm, and the intensity of the major peak was positively correlated with ONOO(-) concentration. Betanin inhibited the ONOO(-) (0.5 mM)-dependent nitration of tyrosine (0.1 mM). Additionally, the IC(50) value of betanin (19.2 μM) was lower than that of ascorbate (79.6 μM). The presence of betanin (0.05-1.0 mM) also inhibited ONOO(-) (0.5 mM)-dependent DNA strand cleavage in a concentration-dependent manner. These results suggest that betalains can protect cells from nitrosative stress in addition to protecting them from oxidative stresses.     

Analysis of betalains from fruits of Opuntia species

Betalains are of great taxonomic significance in higher plants and occur only in 10 families of the order Caryophyllales (Centrospermae). They are water-soluble nitrogenous pigments. They can be divided into two major structural groups, the red to red-violet betacyanins and the yellow betaxanthins. Betalains are widely used as natural red food colorant as well as antioxidant potentials. Several methods have been published for the determination of betalain in fruits of Opuntia species. The purpose of the current review is to provide a systematic survey of the analytical techniques for the determination of betalain from fruits of Opuntia species.     

植物中甜菜色素的研究进展

甜菜色素是一类主要存在于石竹目植物中的天然植物色素，分为甜菜红素和甜菜黄素。甜菜色素不仅具有吸引昆虫授粉的作用，还可以作为一种重要的渗透调节物质和非酶促抗氧化剂来帮助植物抵抗逆境，维持植物体内正常的生理活动。甜菜色素生物合成途径独特，具有重要的医疗保健价值，目前已被广泛用于食品、药物和化妆品中。本文结合国内外最新研究进展，从理化性质、合成途径、生物学功能和应用价值等方面对甜菜色素进行了介绍，重点阐述了甜菜色素和花青素的关系以及光照对甜菜色素生物合成的影响，以期为进一步深入了解甜菜色素和开发利用甜菜色素提供参考依据。     

Establishment and characterization of a betaxanthin-producing cell culture from Portulaca grandiflora

Cell cultures derived from three yellow flowering Portulaca grandiflora genotypes contained betacyanins rather than betaxanthins. A betaxanthin-producing cell culture was obtained by subculturing orange cell clusters isolated from the red-violet cell culture of a violet flowering P. grandiflora genotype. Selection of the most strongly yellow coloured cell material reduced the portion of betacyanins considerably and resulted in a P. grandiflora cell culture characterized by a high concentration of betaxanthins and the occurrence of free betalamic acid. Vulgaxanthin I was the main compound. Besides these pigments carotenoids and flavonoids were detectable.Betaxanthin biosynthesis strongly dependend on light. Product accumulation reached its maximum during the stationary phase of the growth cycle. Excretion of pigments, especially of betalains, could not be detected. Vulgaxanthin I as found in the cell culture was identical with one of two main betaxanthins in the yellow petals of P. grandiflora.The yellow P. grandiflora cells grew well on solid modified Murashige and Skoog medium but failed to grow in liquid medium after a few subcultures. In contrast, white P. grandiflora suspension cultures could be established repeatedly.Abbreviations A absorbance - BX I and BX II betaxanthin fractions - DOPA dihydroxyphenylalanin - DW cell dry weight - molar extinction coefficient - SS solvent system     

Integrated Recovery of Pigments Released from Red Beet Hairy Roots Exposed to Acidic Medium

Hairy root cultures of Beta vulgaris L grown in a bubble column reactor were permeabilised by exposure to B5 medium of pH 2.0. The roots released 39% of their total pigments on a 10 min exposure to B5 medium of pH 2.0 followed by return to standard 135 medium. The pigments released in the extracellular medium were recovered on an adsorption column containing XAD-16 resin. The permeabilised roots regrew and accumulated additional pigments. Comparison of this technique with the previously used techniques like oxygen starvation and temperature shock to permeabilise beet hairy roots suggest that pH mediated release of betalains can be an effective method of releasing betalains from root cultures.